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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 125 F.16 (Poster) Addressing the human HIV-1 reservoir: I. successful C-type lectin-specific liposomal targeting of immature and mature myeloid dendritic cells, macrophages, and blood monocytes Gieseler R.K. 1 , Marquitan G. 2 , Schwarz A. 2 , Streicher H. 3 , Fritze A. 4 , Wader T. 2 , Hahn M. 2 , Schubert R. 4 , Scolaro M.J. 1 1 Rodos BioTarget GmbH, Div. of Research & Development, Hannover, Germany, 2 LTBH Medical Research Institute, Div. of Research & Development, Alta Loma, California, United States of America, 3 University of Sussex, Dept. of Chemistry, Falmer, Brighton, United Kingdom, 4 Albert-Ludwigs University, Dept. of Pharmaceutical Technology and Biopharmaceutics, Freiburg, Germany Objective: Antigen-presenting cells (APCs) express C-type lectins (CTLs) that bind infectious agents, such as HIV, by distinctive sugars. Once internalized, HIV evades the required process of degradation, hence establishing highly infectious long-term viral reservoirs. HAART cannot address nonreplicative virions. We thus aimed at designing a CTL-specific targeting system that may deliver agents for eradicating APC-resident HIV reservoirs. Methods: Calcein-loaded (50 mM) DOPC/Chol/DOPE-MBP (36.5:33.0:2.5 mol:mol:mol) 150–200 nm liposomes were tagged with a cholesterol-linked fucose derivative (Fuc-C4- Chol), or the respective mannose (Man-C4-Chol; pos. ctrl.) or galactose (Gal-C4-Chol; neg. ctrl.) derivatives. Blood monocytes, MO-derived 5-day immature or 7-day mature myeloid dendritic cells, or 7-day macrophages generated under standard conditions were incubated at 5x10E4–2x10E5 cells with 0.1, 1.0, or 10.0 microliters of either of the targeting systems, for 2 or 3 h at 37°C. Results: Fuc-4C-Chol liposomes highly specifically and efficaciously targeted all APCs. Excellent targeting specificity, time-dependent binding, and highly efficacious uptake of tracer dye by all APC types was verified by fluorescence microscopy and flow-cytometry. Besides faint cytoplasmic distribution, calcein most strongly accumulated in the cells’ endosomal/lysosomal system that typically harbors HIV in reservoir cells. Intriguingly, Fuc-4C-Chol liposomes turned out even more efficacious than Man-4C-Chol-labeled pos. ctrls., suggesting increased CTL affinity by favorable steric alteration of the fucosyl residue. Cellular binding of Fuc-4C- Chol- or Man-4C-Chol-targeted liposomes was completely inhibited by 100 mM of soluble L-fucose or D-mannose (pos. ctrl.) [D-galactose (neg. ctrl.) was ineffective], thus clearly demonstrating high CTL specificity. Moreover, these results were achieved in the presence of mannose-binding lectin that, despite being known to prevent CD209 binding of HIV, did not hamper the targeting system’s interaction with membraneexpressed CTLs. Conclusions: Fuc-4C-Chol liposomes may be utilized for directional intracellular delivery of active compounds to CTLexpressing APCs, so as to therapeutically address chronically infected HIV reservoir cell populations F.17 (Poster) Addressing the human HIV-1 reservoir: II. Inactivation of M- and T-tropic HIV-1 strains in myeloid dendritic cells by targeted delivery of the HIV-agglutinating lectin Con A Scolaro M.J. 1 , Schwarz A. 2 , Wader T. 2 , Fritze A. 3 , Schubert R. 3 , Gieseler R.K. 1 1 Rodos BioTarget GmbH, Div. of Research & Development, Hannover, Germany, 2 LTBH Medical Research Institute, Div. of Research & Development, Alta Loma, California, United States of America, 3 Albert-Ludwigs University, Dept. of Pharmaceutical Technology and Biopharmaceutics, Freiburg, Germany Objective: A fucose-dependent (Fuc-4C-Chol) C-type lectin (CTL-)specific targeting system addressing antigen-presenting cells (APCs) (see previous abstract), may be utilized for intracellular endosomal/lysosomal delivery of therapeutic compounds to HIV-1 reservoir cells. We thus inquired whether delivery of an HIV-agglutinating lectin inactivates such infectious HIV-1 reservoirs by employing concanavalin- A (Con-A) that has been shown to most specifically bind alpha-Man-(1-3)-[alpha-Man-(1-6)]-Man (mannose) cores of N- linked oligosaccharides and to potently agglutinate HIV-1. Methods: Fuc-C4-Chol liposomes (see previous abstract) were loaded with horseradish peroxidase-conjugated Con-A (0.5 mg/ml ConA/HRPO). The system was trypsinized to remove payload potentially embedded within, or adhering to, the liposomes’ membranes (bearing the risk to invalidate the targeting mechanism), and the preparations were purified by gel chromatography and kept dark at 4°C until used. Monocyte-derived immature or mature dendritic cells were infected with M-tropic HIV-1 Ada-M (at 67 x TCID50) or T-tropic HIV-1 Lai (at 6.7 x TCID50) on days 2, 4, or 6. One day after infection, thereby allowing reservoir establishment, DCs were treated with lectin-loaded targeting system under the conditions specified in our previous abstract. Results: DC clustering indicating was evaluated on day 8: while homo- and heterotypic clustering is upregulated upon HIV-1 infection, under all conditions upon treatment with the ConA/HRPO-loaded Fuc-C4-Chol targeting system, the clustering behavior of DCs infected with either M- or T-tropic HIV-1 strains was normalized. In addition, HIV-1-dependent increased death rates of DCs were normalized upon treatment. Moreover, the pathologic ratio between veiled-cell and dendritiform DC morphologies upon HIV-1 infection was largely normalized after treatment. Finally, preliminary results revealed cessation of the deleterious transfer of HIV-1 from DCs to T cells, complemented by PCR results verifying the absence of T cell-integrated HIV provirus. Conclusions: By demonstrating elimination of both M- and T-tropic strains of HIV-1, APC-targeted delivery may pave the way for a novel mode of treatment to eventually eliminate chronic intracellular HIV-1 reservoirs.
126 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 F.18 (Poster) The observation of CD4+ T cell stabilization by long term application of low dose prednisolone needs verification in a prospective and controlled clinical trial Stoll M. 1 , Bogner J.R. 2 , Salzberger B. 3 , Stellbrink H.J. 4 , Kasang C. 5 , Brockmeyer N. 6 , Michalik C. 7 , Ulmer A. 8 , Kompetenznetz HIV/AIDS 1 Medizinische Hochschule Hannover, Zentrum Innere Medizin, Abt. Klinische Immunologie, Hannover, Germany, 2 Klinik der Ludwig Maximilians Universität, Med. Poliklinik, Infektionsabteilung, München, Germany, 3 Universitätsklinikum, Klinik I für Innere Medizin, Regensburg, Germany, 4 ICH Hamburg, IPM Study Center, Hamburg, Germany, 5 Missionsärztliches Institut, Würzburg, Germany, 6 Dermatologische Klinik der Ruhr Universität, St. Josef Hospital, Bochum, Germany, 7 Universität Köln, Koordinierungszentrum für klinische Studien (KKSK), Köln, Germany, 8 Praxiszentrum, Stuttgart, Germany Objective: The benefit of HAART is well established. But until now a gold standard for asymptomatic HIV+ individuals without considerable immuno-deficiency is not yet defined. More recently some ongoing studies address the application of HAART in early stages of asymptomatic HIV-infection and immune modulatory proposals with Interleukin-2. In an alternative approach observational studies during the last decade described a slower decay of CD4+ T cells in asymptomatic HIV infected individuals after treatment with corticosteroids. This observation needs verification in a controlled clinical study. The PreTreat study - an investigator-initiated trial had been submitted recently for a public funding by German authorities. Methods: PreTreat is intended as a randomized, doubleblinded, prospective multicenter phase III trial, which will be performed under the auspices of the German Competence Network HIV/AIDS. Antiretroviral naive patients in early stages of HIV disease will receive prednisolone (5 mg/d) or placebo for 96 weeks. Thus the placebo arm is up to the recent standard of treatment from guidelines, which unisonously recommend a “wait and see” strategy in such patients. Inclusion criteria: HIV+, HAART naive, CD4+T cells 400/l (18%), CDC-stage A1, A2 or B1. Exclusion criteria: Need for HAART, CDC-stage B with pVL > 50.000 cp/ml, HbsAg+, relevant comorbidities. Primary endpoint will be the time to any of the following events: • Decay of CD4+ T cells below the threshold of 300/l or < 15% (or < 400/l or 18% for three times consecutively). • Initiation of HAART. • Disease progression or death. Secondary endpoints: Decay of CD4+ T cells, CD4/CD8- ratio, p-VL, tolerability/adverse events. In addition more detailed functional immunological tests will be performed in subgroups. Conclusions: As future prospects the PreTreat study will investigate in summary: (a) whether HAART can be deferred by low dose prednisolone, (b) the effects on surrogate markers of immune response or viral replication, and (c) the toxicity of low dose prednisolone in this setting. The treatment strategy would substitute a period of presently therapeutic nihilism, might reduce long term toxicity of HAART and would be linked with low direct drug costs. F.19 (Vortrag) Development and immunogenicity of RNA- and Codon-optimized HIV candidate vaccines in phase I clinical trials Wagner R. 1 , Wild J. 1 , Bieler K. 2 , Wolf H. 3 , Mooij P. 4 , Heeney J. 4 , Weber J. 5 , Harrari A. 6 , Pantaleo G. 6 1 Universität Regensburg, Institut für Medizinische Mikrobiologie und Hygiene, Molekulare Mikrobiologie und Gentherapie, Regensburg, Germany, 2 Geneart AG, Regensburg, Germany, 3 Universität Regensburg, Institut für Medizinische Mikrobiologie und Hygiene, Regensburg, Germany, 4 BPRC, Rijswijk, Netherlands, 5 Imperial College, London, United Kingdom, 6 CHUV, Lausanne, Switzerland The objective of this study was to design HIV-specific T-cell vaccines comprising DNA- and poxviral vectors and to determine the safety, immunogenicity and efficacy of these candidate vaccines in preclinical and phase 1 clinical trials. Safety and immunogenicity issues were addressed by rational design of RNA and codon-optimized genes encoding a polyprotein comprising Gag, Pol, Nef (GPN) and Env (E) of a clade C HIV isolate that represents the major epidemic in Asia. These expression constructs enabled abundant in vivo expression of HIV proteins in absence of an otherwise nondispensable viral RNA shuttle protein (Rev) and eliminated the risk for recombination between the vaccine and HIV. Established candidate vaccines comprised (i) a DNA vaccine lacking antibiotic resistance genes (DNA- C) and (ii) a replication-deficient vaccinia virus (NYVAC-C), both expressing GPN and E. Following immunological and toxicological analysis in mice, preclinical studies were performed in groups of each 10 rhesus macaques using cGMP materials and matching current clinical protocols. DNA-C/NYVAC-C prime/boost regimens induced substantial antiviral cellular immune responses (ELISPOT, gamma-IFN, IL4, IL6) in rhesus macaques, preserved CD4 cell counts at pre-study levels for more than a year. Following heterologous challenge with pathogenic SHIV89.6P, animals efficiently controlled virus replication and protected animals from disease. Two phase 1 clinical trials involving 40 HIV-negative volunteers demonstrated the vaccine to be safe, well tolerated and immunogenic in humans. About 50% of the vaccinees that received 2 injections of NYVAC-C responded with detectable levels of HIV specific gamma-IFN+ T-cells. Noteworthy, 2 DNA-C priming immunizations (w0, w4) per se induced statistically significant T cell responses in 30% of the vaccines and properly primed T cells. 2 NYVAC booster immunizations (W20, w24) increased the response rate to >90% and significantly enhanced levels of both HIV-specific CD4+ and partially CD8+ T-cells. T cell responses were stable over time, directed against a broad variety of epitopes with some dominance of Env over Gag.
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